Throughout this application various publications are referenced, many referenced by numbers in parenthesis. Full citations for these publications are provided at the end of the Detailed Description of the Invention. The disclosure of these publications are hereby incorporated by reference, in their entirety, in this application.
The invention is in the field of biochemistry and medicine relates to the 24,000 Dalton form of fibroblast growth factor-2 (24 kDa FGF-2). Specifically, this invention relates to any and all portions of the 24 kDa FGF-2 that inhibit the migration of eukaryotic cells but lack the growth promoting activity associated with full length 24 kDa FGF-2.
Polypeptide growth factors stimulate the growth and migration of a variety of cells (1). One of these polypeptide growth factors that promotes endothelial cell growth, migration, and invasion is basic fibroblast growth factor (FGF-2) (2-5). FGF-2 is part of a large family of fibroblast growth factors consisting of at least 9 separate gene products, which share a common domain. The single copy gene for FGF-2 encodes for multiple forms of the protein of 24, 22.5, 22, and 18 kDa with the three higher molecular weight FGF-2s (“hmwFGF-2”) produced by initiation of translation at CUG initiation sites upstream from the AUG codon (FIG. 1) (6; 7). The 24 kDa FGF-2 form is comprised of the 18 kDa FGF-2 with an additional 55 amino acids on the amino terminal end. The structure of the mRNA indicates that its synthesis is translationally controlled. The cellular localization and apparent functions of 18 kDa and hmwFGF-2 differ. The 18 kDa FGF-2 is mostly cytoplasmic and is exported to the cell surface where it is localized to the basement membrane or extracellular matrix in association with matrix heparins and heparans (8; 9). In contrast, undetectable or extremely low levels of hmw FGF-2 are present in the media of the cultured cells studied to date. Instead, the majority of the cellular hmwFGF-2 is directly translocated into the nucleus (10; 11). The residues associated with nuclear translocation are RG repeats found at several sites within the amino terminal region of hmwFGF-2 (12). Thus, 18 kDa FGF-2 has been considered to be an external regulator of endothelial cell behaviour while the hmwFGF-2 is thought to generate intranuclear autocrine signals.
We demonstrated that exogenously applied recombinant 24 kDa FGF-2 could regulate cell behavior in two ways, stimulation of cell proliferation and inhibition of migration (13). The increase in proliferation was comparable to that promoted by 18 kDa FGF-2 indicating that the stimulation was independent of the additional amino terminal peptide. On the other hand, the effect on migration was opposite to that of 18 kDa FGF-2. While 18 kDa FGF-2 promoted cell motility, 24 kDa FGF-2 inhibited migration of endothelial cells by 50% and mammary carcinoma MCF-7 cells by greater than 70%, even in the presence of unrelated mitogens that promote cell migration such as vascular endothelial growth factor (VEGF) and insulin like growth factor-1 (IGF-1). Using antibodies specific to the amino terminal end (amino terminal 55 amino acids, “ATE”) or antibodies to the 18 kDa regions of the 24 kDa FGF-2, we localized the inhibition of migration to the ATE and stimulation of growth to the 18 kDa domain of 24 kDa FGF-2. Thus, it was concluded that 24 kDa FGF-2 affects cell behaviour differently than 18 kDa FGF-2 and that the ATE region, which is absent from the 18 kDa FGF-2, is responsible for this difference.
The present invention is a truncated form of Fibroblast Growth Factor, thus it has never before been described as an independent molecule. The full length Fibroblast Growth Factor has both an inhibitory activity and a proliferative activity, i.e., it stimulates cells to grow which is an unwanted activity in cancer therapy. Growth factors are considered pro-migratory and pro-angiogenic, and they can be used to stimulate angiogenesis in patients with vascular insufficiencies. The unexpected result of the present invention includes the separation of the inhibitory activity from the unwanted proliferative activity, thus allowing the use of a truncated growth factor as anti-angiogenic or an anti-migration compound. The present invention is not an anti-angiogenic alone, but it is effective against tumor cells. Thus, tumors that are not susceptible to anti-angiogenic treatment will be responsive to the present invention.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.